Project Summary/Abstract Viral gene sequences represent a rich and valuable source of information about biological processes. Advances in next generation sequencing (NGS) technology over the past decade now provide the opportunity to probe viral population diversity in unprecedented ways. We recently developed the Primer ID strategy to overcome several serious limitations of conventional NGS including greatly reducing the mis-incorporation and recombination introduced by the preceding PCR step, reducing the errors of the sequencing platform, and revealing sampling depth of the initial viral genomes/templates that are actually represented in the final data set. In this application we describe how we will use Primer ID with NGS to address long-standing issues in HIV-1 population analysis in the context of viral evolution in the absence of therapy, with failed therapy, and with suppressive therapy. In addition, we will link our sequence data to state-of-the-art evolutionary analysis with a goal of making high quality data sets and tools readily available for further secondary analyses. In Aim 1, we will obtain longitudinal plasma samples of 28 HIV-infected women (from the WIHS cohort) starting with high CD4+ T cell counts until they progress to CD4+ T cell counts of less than 100 cells/L. We will perform multiplexed Primer ID sequencing to obtain near full length HIV-1 genome. We predict that X4 variants in viral populations first emerge at low abundance that we will be able to detect much earlier than previously observed. In addition, we will investigate longitudinal viral diversity changes in all sequenced regions to assess population dynamics and link all of these markers to markers of inflammation, CNS damage, and the breadth and potency of neutralizing antibodies. In Aim 2, we will apply the multiplexed Primer ID sequencing approach as a screening tool for drug resistance testing. We hypothesize that the potential for drug resistance mutations to mediate escape can occur from minor variants, too minor to be reliably detected with the methods that have been used to date. We will analyze samples from a large cohort of subjects attending the HIV clinic at the #8 Hospital in Guangzhou, China, to determine how often minor variants are missed by assessing therapy failure using Sanger sequencing, determine the role of transmitted or pre-existing mutations in the failure of WHO first line therapy, and define the mutation pattern after subsequent failure of WHO second line therapy. In Aim 3, we will use deep sequencing with Primer ID to monitor population changes in subjects on therapy. We hypothesize that changes in the low level viral populations occur even while on therapy and that these can be monitored using deep. The insights gained will be informative for how to apply this technology to search for evolutionary variants related to X4 viruses, to define other biological correlates of the changing viral population with disease progression in women, to define how the viral population gets established with regard to the steady-state levels of minor sequence variants, to examine the role of drug resistance variants in therapy failure, and to explore the utility of NGS technology in the setting of subjects on therapy.